This invention relates to the field of polynucleotides and polypeptides. More specifically, this invention relates to TUP1 polynucleotides from Candida albicans, Tup1 polypeptides, and methods using these polynucleotides and polypeptides, especially for screening candidate anti-fungal agents.
The yeast Candida is a ubiquitous human commensal, known as the causative agent of candidiasis. The majority of the diseases are caused by the species Candida albicans. It is the most prevalent commensal and opportunistic fungal pathogen of humans, causing common superficial infections as well as more serious systemic and organ infections. Cannon et al. (1995) J. Dental Research. 74:1152-1161. Exposure to C. albicans at or shortly after birth results in lifelong colonization in the host tissues, such as the gastrointestinal tract, oral cavity and genital area. It has been noted that approximately 75% of women would suffer from vaginal candidiasis at some stage in their lifetime. Bossche et al. (1993) Fungal Dimorphism 3-10; Fidel et al. (1996) Clin. Micro. Rev. 9(3):335-348. Whereas C. albicans infection often remains localized to the initial sites of contact in healthy individuals, C. albicans cells can invade submucosal vessels, disseminate hematogenously and become life-threatening, especially to immunocompromised patients. The invasive forms of C. albicans infection are not only dangerous in their own right, but they are believed to facilitate infections by other opportunistic pathogens.
In the last decades, the incidence of severe and systemic candidiasis has increased dramatically because of the growing number of immunocompromised patients suffering from AIDS, diabetes, cancer and other conditions. In addition, the widespread use of immunosuppressants for organ transplant patients, the common practice of radiation and chemotherapy for treating malignancies, as well as the growing size of the aging population have increased the morbidity of this opportunistic pathogen. For reviews, see Rubin et al. (1993) Eur. J Clin. Microbiol. Infect. Dis. 12 Suppl. 1, 542; Dudley et al. (1990) Pharmacotherapy 10:133; Paya (1993) Clin. Infect. Dis. 16:677-688; Rubin (1993) Eur. J Clin. Micro. Infect. Dis. 12 Suppl. 1: S42-S48.
Despite decades of intensive study, the properties of C. albicans that contribute to its virulence are only beginning to be understood. Among the most investigated virulence factors are adherence, production of hydrolytic enzymes and adoption of various cell morphologies. Odds et al. (1994) Am. Soc. Microbiol. News 60:313-318. The ability of C. albicans to adhere to the host surfaces probably allows initial colonization and infection of the host tissues. Secretion of a variety of hydrolytic enzymes which are capable of degrading proteins and lipids is thought to generate tissue cavitation and thereby facilitate deeper penetration. The morphological transition between various forms of C. albicans is also considered a key determinant of virulence.
C. albicans cells can exist in a variety of shapes, ranging from elipsoidal budding yeast cells (also known as blastopores) to cylindrical hyphae (also known as filaments) in which cells remain attached to each other after dividing and thereby form long branched strings of connected cells (FIG. 1). Transitions between these forms take place by outgrowth of new cells with the altered morphology, rather than remodeling of pre-existing cells. The ability of C. albicans to adopt these different morphologies is thought to allow the fungus to adapt to, and possibly travel to, different host micro-environments. Odds et al. (1988) Candida and Candidosis (Bailliere Tindall, London, 2nd ed.); Odds et al. (1994); Odds et al. (1994) J. Am. Acad. Dermatol. 31:52. The regulation of cellular morphology is in response to environmental conditions. In vitro studies have shown that most C. albicans strains assume filamentous forms when they are subjected to either unfavorable growth conditions, such as nutrient-poor media and high CO2:O2 ratio, or host-related conditions, such as high temperature (37xc2x0 C.) and mammalian serum (10%). Conversely, rich media, low temperatures and aerated conditions promote blastospore growth. Intermediate conditions can induce various pseudohyphal forms as well as true hyphae. For reviews, see Odds et al. (1988) Candida and Candidosis, Bailliere Tindall, London, ed. 2nd; Odds et al Crit. Rev Microbiol. (1985) 12:45; Gow et al. (1984) Sabouraudia 22:137. The pseudohyphal cells are elongated but still elipsoidal in shape, whereas the true hyphal cells are cylindrical and separated by perpendicular septal walls. Very little is known about the genetic identity of regulators controlling the morphological transition of C. albicans. 
The ability of C. albicans to adopt these different morphologies is thought to contribute to colonization and dissemination within host tissues and thereby to promote infection. Odds (1988); Odds (1994) J. Am. Acad. Dermatol. 31:S2. It has been commonly suggested that the hyphal form is invasive and pathogenic, while the blastospore is the commensal non-pathogenic form. However, all morphological forms have been found within infected tissues. Histopathological examination of candidiasis lesions indicates that hyphae are not always present. More recent studies have shown that commensal C. albicans does not exist uniquely in the blastopore form. In fact, sometimes invading C. albicans cells are seen exclusively as the budding yeast form. Odds et al. (1994) Am. Soc. Microbiol. News 60:313-318. Despite the uncertainty with regard to the relative roles these two distinct forms of C. albicans have in fungal virulence, phenotypic switching represents a remarkable adaptation that C. albicans has acquired to cope with different host microenvironments. Identifying the genetic components that regulate the morphological transition are therefore of great significance for identifying the role of this transition in pathogenesis and developing potential therapeutic agents of candidiasis.
Current therapy available for systemic candidiasis is limited to the use of anti-fungal agents. In practice, the arsenal of anti-fungal drugs is based on a few antimycotics, such as flucytosine, amphotericin B and azole derivatives. Many of these antimycotics are somewhat water insoluble which restrict their bioavailability and present problems in intravenous formulation. In addition, they cause serious and often difficult side effects, such as renal toxicity, bone marrow destruction, as well as unpleasant symptoms such as fever and shivering. Furthermore, the chronic use of these anti-fungal agents has led to the emergence of drug-resistant strains of Candida, which can cause fatal relapse of the disease. Dupont et al. (1995) J. Am. Podiatric Med. Assn. 85:104-115; Fox et al. (1991) J. Infect. Dis. 22:201-204; Scheife (1990) Pharmacotherapy 10:S133-S183. Taken together, anti-fungal therapy alone is inadequate for treating chronic candiasis. The availability of recombinant cytokines, such as interleukin-2, provides an alternative way to stimulate the cell-mediated immunity of infected individuals. However, this type of cytokine replacement therapy for fungal infections remains highly experimental. Weinberg et al. (1990) N. Eng. J. Med. 332: 1718.
S. cerevisiae Tup1 encoded by the TUP1 gene is a member of a family of WD repeat containing proteins. Tup1, along with the SSN6 protein, represses sets of genes involved in a variety of cellular processes, including glucose repression, mating, sporulation and flocculation. The gene targets of TUP1 regulation are each regulated by a distinct upstream DNA-binding protein, and each DNA-binding protein recruits to the promoter a complex containing the TUP1 protein. The biochemical mechanisms by which TUP1 in S. cerevisiae mediates transcriptional repression are yet not well understood. Tzamarias et al. (1994) Nature 369: 758; Komachi et al. Genes Dev. 8: 2857; Wahi et al. (1995) Genetics 140: 79-90; Edmondson et al. (1996) Genes Dev. 10: 1247.
In addition to the S. cerevisiae TUP1 gene, TUP1 homologs in higher eukaryotes such as human, mouse and chicken have been identified and found to be highly diverged from S. cerevisiae TUP1. Virtually nothing is known about the biological functions of these genes. The genomic organization of the human TUP1, however, has been characterized. It is located on chromosome 22q11, within the Digeorge syndrome critical region. Hemizygosity of this region results in DiGeorge syndrome, which is a developmental disorder characterized by aplasia or hypoplasia of the thymus and parathyroid glands, as well as conotruncal cardiac malformations. Llevadot et al. (1996) Mammalian Genome 7: 911-914.
In view of the alarming prevalence of life-threatening candidiasis among immunocompromised patients and the lack of satisfactory agents to treat this condition, there is a pressing need for developing better therapeutic agents to combat C. albicans infections.
All publications cited herein are hereby incorporated by reference in their entirety.
This invention provides C. albicans TUP1 gene polynucleotide sequences, Tup1 polypeptides encoded by these sequences, antibodies that bind to these polypeptides, compositions comprising any of the above, as well as methods using the polynucleotides, polypeptides, and/or antibodies.
Accordingly, in one aspect, the invention includes an isolated polynucleotide comprising a sequence encoding a Tup1 polypeptide from C. albicans, wherein the polypeptide complements a tup1 mutation in a yeast cell. The Tup1 polypeptide encoded is found within the sequence depicted in SEQ ID NO:2, including from about 190 to about 465, about 1 to about 465, about 1 to about 512 of SEQ ID NO:2, as well as the entire sequence of SEQ ID NO:2.
In another aspect, the invention provides isolated polynucleotides based on the sequence depicted in SEQ ID NO:1, and as such may comprise nucleotides from about 904 to about 1728, from about 354 to about 1728 of SEQ ID NO:1, as well as the entire sequence of SEQ ID NO:1.
In another aspect, the invention provides an isolated polynucleotide comprising the polynucleotide sequence of SEQ ID NO:1. In another aspect, the isolated polynucleotide comprises a region of at least 20 contiguous nucleotides, with the region having at least 80%, preferably at least 85%, sequence identity with a sequence depicted in SEQ ID NO:1. In other embodiments, the invention provides an isolated polynucleotide comprising a region of at least 20 contiguous nucleotides, with the region (and/or isolated polypeptide comprsing this region) able to hybridize under stringent conditions to a sequence depicted in SEQ ID NO:1, particularly SEQ ID NO:1.
In another aspect, the invention includes cloning vectors, expression vectors, host cells, and compositions comprising any of the above polynucleotides.
In another aspect, the invention provides an isolated polypeptide comprising a Tup1 polypeptide sequence from C. albicans, wherein the polypeptide complements a tup1 mutation in a yeast cell. In one embodiment, the polypeptide comprises about amino acid 190 to about 465 of SEQ ID NO:2. In another embodiment, the polypeptide comprises about amino acid 1 to about amino acid 465 of SEQ ID NO:2. In another embodiment, the polypeptide comprises the sequence of SEQ ID NO:2.
In another aspect, the invention includes compositions comprising any of the polypeptides of the invention.
In another aspect, the invention provides purified antibodies that are capable of specifically binding to a polypeptide of the invention. In another aspect, the invention provides a monoclonal antibody capable of specifically binding to a polypeptide of the invention.
In another aspect, the invention provides an isolated C. albicans cell having compromised TUP1 function.
In another aspect, the invention provides a method for detecting a polynucleotide from C. albicans in a sample comprising the steps of (a) contacting polynucleotide from C. albicans from a sample with a polynucleotide of this invention under conditions that permit the formation of a stable duplex; and (b) detecting the stable duplex formed in step (a), if any.
The invention also provides a method for detecting a polynucleotide from C. albicans in a sample comprising the steps of (a) conducting an amplification reaction on a polynucleotide in the sample using a primer consisting of a fragment of the polynucleotide sequence of SEQ ID NO:2; and (b) detecting the presence of amplified copies of the polynucleotide, if any.
The invention also provides a method for detecting an anti-C. albicans Tup1 antibody in a biological sample, comprising the steps of: (a) contacting antibody from the sample with a polypeptide of this invention under conditions which permit formation of a stable antigen-antibody complex; and (b) detecting said stable complexes formed in step (a), if any.
The invention also provides a method for detecting a C. albicans Tup1 polypeptide in a biological sample, comprising the steps of: (a) contacting polypeptide from the biological sample with an antibody of this invention under conditions that permit the formation of a stable antigen-antibody complex; and (b) detecting said stable complexes formed in step (a), if any.
In another aspect, the invention provides methods for identifying an agent that may control virulence in C. albicans. These methods may be in vitro or in vivo (i.e., cell-based). In one embodiment, the invention provides a method for identifying an agent that may control virulence in C. albicans, said method comprising:
(a) contacting at least one agent to be tested with a suitable host cell that has TUP1 function;
(b) analyzing at least one characteristic which is associated with loss of TUP1 function in said host cell, wherein an agent is identified by its ability to elicit at least one such characteristic.
In another embodiment, the invention provides a method for identifying an agent that may control virulence in C. albicans, said method comprising:
(a) introducing a polynucleotide encoding C. albicans Tup1 or a functional fragment thereof into a suitable host cell that otherwise lacks TUP1 function, wherein TUP1 function is restored in said host cell;
(b) contacting said host cell of step (a) with at least one agent to be tested;
(c) analyze at least one characteristic which is associated with loss of TUP1 function in said host cell, wherein an agent is identified by its ability to elicit at least one such characteristic.
In another aspect, the invention provides compositions for controlling virulence in C. albicans comprising any agent identified by the screening methods above.
In another aspect, the invention provides kits for detection or quantification of (a) a polynucleotide comprising a TUP1 polynucleotide from C. albicans; or (b) a C. albicans polypeptide; or (c) an anti-C. albicans antibody in a biological sample. These kits contain (a) a polynucleotide of the invention; or (b) an antibody of the invention; or (c) a polypeptide of the invention, respectively.
In another aspect, the invention provides methods of inhibiting virulence of C. albicans comprising compromising C. albicans TUP1 function.
In another aspect, the invention provides a method of isolating a polynucleotide sequence from C. albicans that is associated with C. albicans TUP1 function, said method comprising identifying a transcribed polynucleotide which is repressed upon C. albicans TUP1 expression.